Biopsy support with sectionable resilient cellular material

ABSTRACT

A histologic tissue sample support device includes a tissue support formed of material that can be successfully sectioned in a microtome and is resistant to degradation from solvents and chemicals used to fix, process and stain tissue. A resilient cellular material is coupled to the tissue support and is configured to engage and retain tissue in place during processing and embedding. The resilient cellular material is also capable of successful sectioning in the microtome and porous to allow infiltration of the solvents and chemicals used to fix, process and stain tissue, and of embedding material used to embed the tissue while the tissue is retained by the resilient cellular material.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/869,629, filed Dec. 12, 2006, the disclosure ofwhich is also fully incorporated by reference.

TECHNICAL FIELD

The present invention generally relates to supports for handling andembedding tissue samples for pathological analysis and, moreparticularly, to sectionable supports which can receive one or moretissue samples and be embedded and subsequently microtomed with thetissue sample or samples.

BACKGROUND

To accurately diagnose various tissue diseases and conditions, medicalpersonnel must remove one or more samples of tissue from the body of apatient. This process of harvesting tissue from the body is known as abiopsy. Once the tissue sample or samples are removed and sent to apathology laboratory, the tissue will go through a series of proceduresperformed by a histotechnician and, ultimately, a pathologist, in orderto diagnose one or more conditions associated with the tissue. Thepresent invention generally relates to those procedures that arenormally performed by the histotechnician to prepare the tissue sampleor samples into slides that may be analyzed under a microscope by thepathologist.

Although the singular term “sample” is used throughout thisspecification, it should be understood that this term likewiseencompasses plural “samples” as well. Once a tissue sample is removedfrom the body of a patient, it is typically placed into a specimencontainer containing a tissue fixative solution and then the containeris transported to a pathology laboratory. The tissue will undergo aprocess known as “grossing-in” in the pathology lab during which ahistotechnician will retrieve the tissue sample from the container,typically cut the tissue into appropriate sizes for tissue processing,place individual samples into the appropriate sized small plastic tissuecassettes, and assign tracking numbers to each cassette. These trackingnumbers are then logged into a tracking system used in the laboratory.For the smallest tissue samples, which may only be scrapings, thecassette includes fine mesh openings on the sides and bottoms. In othersituations involving very small tissue samples, the samples are placedinto a bag that resembles a tea bag that prevents the smallest tissuesamples from escaping. Larger tissue samples are placed into cassetteshaving somewhat larger slotted openings which are nevertheless smallerthan the tissue sample inside the cassette.

The cassettes are then placed into a stainless steel perforated basketand run through a tissue processing machine, often overnight. Thismachine uses a combination of vacuum, heat, and chemicals to remove theinterstitial fluids within the tissue. Once the fluids have been removedfrom the tissue samples, the processing machine immerses the tissuessamples in a bath of a hardenable material such as molten paraffin(i.e., a form of wax) so that the interstices in the tissue are replacedwith paraffin. The histotechnician then removes the basket from themachine and removes the individual tissue cassettes. In a conventionalprocedure practiced for many years, the histotechnician individuallyremoves the tissue sample from each cassette. The histotechnician mustcarefully orient the tissue sample, based on tissue type, into astainless steel base mold that is roughly the size of the tissuecassette and is partially filled with molten paraffin. The tissue samplemust be manually held, typically using forceps, against the bottom ofthe mold. If it is not, this could compromise the ability to make properslices of the tissue sample later in a microtome. The molten paraffin isthen rapidly cooled on a refrigerated plate, which may be a thermalelectric cooler (TEC), to partially solidify the paraffin therebyholding the tissue sample in the proper orientation against the bottomof the mold. The cassette is then placed on top of the base mold and anembedding material, which is also typically paraffin wax, is pouredthrough the opened top of the cassette into the base mold. The cassettechanges its function at this point in the procedure from a tissueholding component to a fixture type device for mounting in the microtomeand making shavings or slices from the solidified paraffin in themicrotome. The base mold is chilled until all of the molten paraffin hashardened and the histotechnician removes the stainless steel base moldfrom the block of embedded paraffin. The tissue sample is thus embeddedwithin a rectangular block of hard paraffin with a plastic tissuecassette on the opposite side. As mentioned, the cassette may then beused as a holder or fixture in the chuck of the microtome. As with thetissue processing machine, the embedding process is accomplished in abatch fashion during which an average histotechnician may embedapproximately 40 to 60 cassettes per hour.

The blocks of hardened paraffin containing the embedded tissue samplesare then ready to be sliced into extremely thin sections for placementon a microscope slide. The histotechnician mounts the embedded tissueblock in a chuck on the microtome that is sized to accept the side ofthe block that has the embedded plastic cassette. The histotechniciancan then begin slicing the paraffin block which has the tissue sampleembedded opposite to the plastic cassette surface. This yields a ribbonof individual slices of the tissue embedded in the hardened paraffin.The action of the microtome causes the individual slices to sticktogether when done properly and, subsequently, these very thin ribbonsof slices are floated into a water bath and a glass slide is carefullyplaced underneath the slice. The slice, with the thin sectioned tissuesample embedded therein, is then adhered to the top of the slide.

When the histotechnician has enough slides from the tissue sample, theslides are placed into an automatic staining machine. The stainingmachine goes through a series of infiltrating steps to stain thedifferent tissue and cells of the slide different colors. This helps thepathologist identify different structures and makes it easier to findany abnormalities in the tissue. After the staining procedure iscomplete, the slides are cover slipped and prepared for the pathologistto place under a microscope for analysis.

Based on the summary of the procedure provided above, it will beappreciated that conventional tissue sample handling and processing is avery labor-intensive process involving several manual steps performed bya histotechnician. Thus, repetitive stress injuries such as carpaltunnel syndrome are prevalent. This is especially true with the tissuesample embedding process. These multiple manual operations and repeatedtissue handling increase the likelihood of human error and, moreover,require highly trained and skilled histotechnicians to ensure that thetissue samples ultimately adhered to the slides for analysis by thepathologist are in an optimum condition and orientation to make accuratediagnoses.

U.S. Pat. Nos. 5,817,032 (the '032 patent) and 7,156,814, and U.S.Patent Application Publication Nos. 2005/0226770; 2005/0147538; and2005/0084425 disclose various improvements to this area of technology,including new manners of holding tissue samples during the grossing in,embedding, and microtome or slicing procedures. The disclosures of U.S.Pat. No. 5,817,032 (the '032 patent) and U.S. Patent ApplicationPublication Nos. 2005/0226770; 2005/0147538; and 2005/0084425 are herebyfully incorporated by reference herein. For example, the '032 patentrelates to a tissue trapping and supporting device, which may be acassette, and which may be successfully sectioned using a microtome.When such a cassette is used, the tissue sample is immobilized withinthe cassette and subjected to the process for replacing tissue fluidswith paraffin. Then, the tissue sample and the cassette are sliced atthe same time for later mounting on microscope slides. Because thetissue sample is never removed from the cassette from the time it isprocessed in the tissue processing machine to the time that it is cut orsliced with the microtome, a significant amount of handling time issaved. Moreover, the chance for human error or tissue loss issignificantly reduced due to the elimination of separate tissue handlingsteps. The '032 patent and the above-incorporated published applicationsalso generally disclose further improvements that help to automate theoverall process and, in conjunction with the novel tissue supports(e.g., cassettes), can even further reduce the handling steps during theentire procedure and make the procedure more reliable.

In spite of the various advances made in this field, there is anincreasing need for additional improvements related to increasedproduction capability and more consistent quality of embedded tissuesamples and resulting slices or ribbons of embedded tissue that will besubject to diagnosis. This can be especially important when handlingsmaller tissue sample sizes, although the improvements to be disclosedherein are applicable to all tissue sample sizes.

SUMMARY

In one general embodiment, a histologic tissue sample support isprovided and may generally comprise a tissue support coupled with aresilient cellular material. The resilient cellular material is a threedimensional, microtome sectionable, deflectable structure that is animprovement upon the microtome sectionable, deflectable structuresdisclosed in the above-incorporated '032 patent discussed above. Thetissue sample support device can more specifically include a tissuesupport formed of material which can be successfully sectioned in amicrotome and is resistant to degradation from solvents and chemicalsused to fix, process and stain tissue. The porosity of the resilientcellular material allows infiltration of the solvents and chemicals usedto fix, process and stain tissue, and of embedding material used toembed the tissue while the tissue is retained by the resilient cellularmaterial. The resilient cellular material has a thickness that iscompressible and configured to engage and retain tissue in place duringprocessing and embedding and is also capable of successful sectioning inthe microtome after having its interstices or pores filled withliquefied embedding material which subsequently hardens.

The resilient cellular material may further comprise an open cell foammaterial, such as a foam including at least one of a polyether or apolyurethane. In addition, the open cell foam may be a fully reticulatedfoam. This helps ensure full infiltration of fluids used duringprocessing and embedding procedures. Other synthetic and naturalmaterials may be used such as polyesters, alginates, or other materialsthat may be infiltrated with the embedding material and successfullysectioned and a microtome without adverse effects on the resultingribbon of tissue and embedding material.

The support may further include a tissue containment portion including arecess or interior area surrounded by at least one side wall andincluding a bottom wall. The recess or interior area may be configuredto at least partially contain the resilient cellular material eitherduring the manufacturing of the device or during insertion of thecellular material into the recess or interior area by the user in orderto retain the tissue sample in place during processing and embeddingprocedures. The support can further comprise a cassette having a lidconfigured to be connected to the containment portion. In oneembodiment, the resilient cellular material is coupled to the lid and isinserted at least partially into the recess upon connecting the lid tothe containment portion.

The material forming the support may be at least translucent so as to benon-distracting during tissue analysis. For example, the support may beformed of any of the materials disclosed in the above incorporatedpatent and patent applications such as polymers including fluorinatedpolymers or fluoropolymers (e.g., PFA).

An assembly may be constructed with the support and a separate frame. Insuch an assembly, the tissue support is releasably retained on the frameand the frame is further configured for releasable securement within amicrotome chuck. The frame can further include an interior and thetissue support may be sized to fit and move within the interior betweenat least a first position and a second position. The first position isused during processing of the tissue sample, and the second position isused to expose the tissue outward of the frame in a position forallowing the tissue sample to be sectioned in the microtome.

Various methods are disclosed or will be apparent based on a review ofthe disclosed embodiments and features. For example, a method forpreparing one or more biopsy tissue samples for histological examinationmay comprise:

-   -   positioning a tissue sample in close proximity to a microtome        sectionable support;    -   immobilizing the tissue sample on a the support by contacting        the tissue sample with a microtome sectionable, resilient        cellular material coupled to the tissue support;    -   subjecting the microtome sectionable support, resilient cellular        material and the tissue sample to a process that replaces fluid        in the tissue sample with a hardenable material;    -   embedding the microtome sectionable support, resilient cellular        material and the tissue sample in an embedding material;    -   hardening the embedding material into a block; and    -   slicing the block with a microtome into thin slices of the        embedding material, the microtome sectionable support, the        resilient cellular material and the tissue sample.

The hardenable material and the embedding material may be the samematerial, such as a wax (e.g., paraffin). The support may furthercomprise a bottom portion configured to hold the tissue sample and a lidholding the resilient cellular material. The step of immobilizing thetissue sample can further comprise closing the lid on top of the tissuesample to trap the tissue sample between the resilient cellular materialand the bottom portion. The bottom portion can include an interior spacesurrounded by at least one side wall and the positioning andimmobilizing steps and can further comprise placing the tissue samplewithin the interior space, and inserting the resilient cellular materialat least partially into the interior space and into contact with thetissue sample. The resilient cellular material may deform during theimmobilizing step to create a three dimensional space that receives thetissue sample. This can help immobilize the tissue sample in a desiredform flat against the bottom of the support or cassette. The force ofthe resilient cellular material against the tissue should be enough toimmobilize and/or flatten the tissue but not enough to induce artifactsin the sample. The microtome sectionable support may be coupled to aframe prior to being subjected to the process for replacing fluid in thetissue sample with the hardenable material. The method can then furthercomprise securing the frame in the microtome prior to slicing the block.Prior to embedding the microtome sectionable support, resilient cellularmaterial and the tissue sample in the embedding material, the microtomesectionable support may be moved from a first position within the frameto a second position in which the support, resilient cellular materialand tissue sample are exposed for simultaneous sectioning in themicrotome.

Various additional details, features, advantages and aspects of theinvention will become more readily apparent to those of ordinary skillin the art on review of the following illustrative, more detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an assembly comprised of a microtomesectionable tissue cassette received within a frame, with the lid of thecassette shown in an open position.

FIG. 2 is a perspective view similar to FIG. 1, but illustrating the lidof the cassette in a closed position.

FIG. 3 is a cross sectional view generally taken along line 3-3 of FIG.2.

FIG. 4 is a cross sectional view similar to FIG. 3, but showing thecassette/frame assembly embedded in paraffin and with the cassette in asecond, lower position within the frame for sectioning.

FIG. 5 is a cross sectional view taken generally along line 5-5 of FIG.4.

FIG. 6 is a cross sectional view taken generally along line 6-6 of FIG.4.

FIG. 7 is a perspective view of an assembly comprised of a microtomesectionable tissue cassette constructed according to another embodiment,and received within a frame, with the lid of the cassette shown in anopen position.

FIG. 8 is a top view of the assembly shown in FIG. 7, but with the lidin a closed position, and the assembly in a mold.

FIG. 9 is a cross sectional view taken along line 9-9 of FIG. 8 andschematically illustrating the lid being depressed into a closedposition.

FIG. 10 is a cross sectional view similar to FIG. 9, but illustratingthe cassette in a second, lower position within the frame such that alower portion of the cassette extends into the mold.

DETAILED DESCRIPTION

FIGS. 1 and 2 generally illustrate an assembly 10 comprised of a tissuesample cassette 12 carried within a frame 14. The connection of thetissue cassette 12 to the frame 14 may be accomplished in many differentmanners, such as any of the manners described in the above-incorporatedpatent and patent applications. It will be also be appreciated that thecassette 12 may be configured in any suitable manner as a tissue supportand the frame 14 may be configured in any suitable manner. Any of theconfigurations, features, characteristics and materials disclosed forthe tissue supports (e.g., cassettes) and frames in theabove-incorporated patent and patent applications may be employed forcassette 12 and frame 14. In the embodiment shown, the cassette 12 isporous and is releasably retained in the frame 14 and the frame 14 isfurther configured to be releasably secured within a microtome chuck(not shown). The frame 14 generally includes an interior defined betweensurrounding outer walls 14 a, 14 b, 14 c, 14 d and the cassette 12 issized and configured to frictionally or “snap” fit and move within theinterior between at least first and second positions, again, asgenerally discussed in the above-incorporated patent and patentapplications and for the same purposes. The first position is shown inFIG. 3, while the second position is a position (not shown) in which thelower portion of the cassette 12 is exposed below the bottom of theframe 14, as viewed in FIG. 3, for allowing the cassette 12 and tissuesample to be sectioned in a microtome while the frame 14 is held in themicrotome chuck. The general procedure for processing, embedding, andsectioning is discussed in the above-incorporated patent and patentapplications. The cassette may be formed from perfluoroalkoxyethylene(PFA) in accordance with the above-incorporated patents and patentapplications.

A lid 12 a of the cassette 12 may be coupled to a body 12 b of thecassette 12 by a hinge 16. The lid 12 a may also snap fit into a closedposition as shown in FIG. 2 through engagement of fingers or projectingconnectors 13 on the cassette body 12 b with an outer flange 15 of thelid 12 a on each of the four sides of the lid 12 a. The lid 12 a carriesa resilient cellular material 20 which may, for example, be an open cellfoam material, such as a foam including at least one of a polyether or apolyurethane and which may be a fully reticulated foam. Here, “fullyreticulated” means that at least substantially all cells of the foam areopen. As shown in FIG. 1, one or more tissue samples may be placed in aporous tissue containment portion 30 that may define a recess orinterior area surrounded by at least one sidewall 32 and including abottom wall 34. Although a circular recess is shown, it will beappreciated that any other shape may be used instead.

As further shown in FIG. 3, when the lid 12 a is closed, the foammaterial 20 will press against the tissue samples 40 and deform threedimensionally around the tissue samples 40 creating three dimensionalspaces around each tissue sample 40 and essentially immobilizing eachtissue sample 40 during the tissue processing and embedding procedures.This also ensures that the tissue samples 40 are held flat against thebottom wall 34 of the tissue cassette 12 such that when microtome slicesare made, complete and continuous sections of the tissue sample 40 maybe formed generally as shown in FIG. 5. One specific type of foamstructure suitable for the resilient cellular material 20 has a poresize of 50-60 ppi (pores per inch), with each pore having a diameter ofbetween about 0.017 inch and 0.20 inch. The foam structure is fullyreticulated with a compression force deflection at 20% deflection of0.55 lbs./in² and a density of 1.4 lbs./ft³. The foam material may beobtained from Crest Foam of Moonachie, N.J. under the name T-50. This isa polyether/polyurethane foam and operates well with a thickness of 0.06inch to 0.10 inch with a 0.075 inch thickness being a practicalmanufacturing example. The foam should be constructed so as to shed orrelease processing fluid after each reagent cycle of a tissue processingmachine. If the foam is too dense or too thick, or not fullyreticulated, the reagents can become cross contaminated or the tissuemay not be fully infiltrated with the fluids because each fluid bathmust fully clear and exchange from one fluid bath to the next.

In use, one or more tissue samples 40 are placed within the interiorspace or recess and, specifically, on the bottom wall 34 as shown inFIG. 1. The cassette lid 12 a is then closed and snapped into place suchthat the resilient cellular material (e.g., foam) 20 bears against andtraps the tissue samples 40 against the bottom wall 34 as shown in FIG.3. At this point, the assembly 10 with the trapped tissue samples 40 maybe subjected to a conventional tissue processing operation that usesvacuum, heat and chemicals to remove the interstitial fluids within thetissue and replace those fluids with a hardenable material, such asmolten paraffin. As mentioned above, during these processing steps, theporous nature of the foam or other resilient cellular material 20 allowsthe fluids to reach and fully infiltrate into the tissue samples 40. Inaddition, the foam 20 traps the tissue samples 40 flat against thebottom wall 34 without leaving artifacts or markings on the tissue thatmight interfere with subsequent analysis under a microscope. It will beappreciated that different types of resilient cellular materials may bechosen based, for example, on the type of tissue to be processed andanalyzed. For example, small mucosal tissue samples may be held andprocessed with success using the T-50 foam discussed above, while othertypes of tissue, such as fatty tissue, may be better served by anothertype of resilient cellular material.

It will also be appreciated that the processing steps may take placebefore assembling the tissue cassette 12 with the frame 14. After thetissue processing is complete, the tissue cassette 12 may be moved to asecond position as shown in FIG. 4 exposing the containment portion 30below the bottom surface 14 e of the frame 14. The cassette 12 and frame14 are then placed into a suitable mold (not shown) and embedded inparaffin 50, such that the entire assembly including the lower exposedcontainment portion 30 are embedded within a hardened block of paraffinwax 50. The mold may generally follow the contour of the bottom of thecassette 12, although the portion of the mold surrounding thecontainment portion 30 is preferably square as opposed to round. Thisassists with the subsequent production of ribbon slices. This portion ofthe procedure may therefore be similar to that disclosed in theabove-incorporated patent and patent applications. As discussed therein,the frame 14 is then used as a fixture for mounting the embeddedassembly 10 in a microtome chuck and the necessary number of slices aretaken of the exposed underside until enough sections, similar to thoseshown in FIG. 5, are taken and appropriately mounted on a microscopeslide, stained and cover slipped.

FIGS. 7-10 illustrate an alternative embodiment. In this embodiment, thecassette is constructed with a somewhat different design than the firstembodiment as will be apparent from a review of the figures, as well asthe description below. Like reference numerals in FIGS. 1-10 refer tolike structure and, therefore, additional description with respect toFIGS. 7-10 is not necessary. Like reference numerals in FIGS. 7-10having prime marks (′) refer to analogous elements as shown anddescribed in connection with FIGS. 1-6 but having differences that areeither apparent by reviewing the drawings themselves or by a combinationof reviewing the drawings and the additional description containedbelow.

The primary difference between assembly 10 and assembly 10′ is withrespect to the lids 12 a, 12 a′ and the manner that the lids 12 a, 12 a′connect with the bodies 12 b, 12 b′ of the cassettes 12, 12′. In thefirst embodiment, the lid 12 a is held down to the body 12 b by a seriesof snap fit connectors 13 on each of the four sides of the cassette body12 b. These connectors 13 engage an outer flange section 15 of the lid12 a. Thus, the user typically would use his or her finger to depresseach of the four sides of the cassette lid 12 a downward to engage eachof the sets of snap fit connectors 13. The cassette 12′ shown in FIGS.7-10 instead uses a circular lid 12 a′ having three connecting elements60 spaced apart by 120°. Elements 60 connect in a snap fit manner withconnectors 62 on the cassette body 12 b′ when the lid 12 a′ is foldedover as shown in FIGS. 8 and 9. As further illustrated in FIG. 9, thelid 12 a′ may be closed and locked in a snap fit manner utilizing thethree mating connectors 60, 62 by depressing the lid 12 a′ with a singlefinger 64 of the user. As previously discussed, the cassette 12′ may bedepressed entirely as a unit from the upper position in the frame 14,illustrated in FIG. 9, to the lower position shown in FIG. 10. Thisextends the porous tissue containment portion 30 into the mold 68. Themold may then be filled with embedding material such as paraffin 50. Astructure is thereby formed as previously described in connection withFIG. 4. It will further be appreciated that the basic perforated designof the cassette 12′ is changed relative to the perforated design of thecassette 12 illustrated in FIGS. 1-6. Essentially, the design of themolded cassette 12′ illustrated in FIGS. 7-10 reduces the amount of PFAmaterial necessary to form the cassette 12′. All other structural andfunctional aspects and uses of assembly 10′ are as described inconnection with assembly 10 of the first embodiment.

While the present invention has been illustrated by a description ofvarious illustrative embodiments and while these embodiments have beendescribed in some detail, it is not the intention of the Applicants torestrict or in any way limit the scope of the appended claims to suchdetail. Additional advantages and modifications will readily appear tothose skilled in the art. The various features of the invention may beused alone or any combinations depending on the needs and preferences ofthe user. However, the invention itself should only be defined by theappended claims.

1. A histologic tissue sample support device comprising: a tissuecassette, the tissue cassette including a tissue containment portionhaving a recess surrounded by at least one side wall and including abottom wall, the tissue containment portion formed of material which canbe successfully sectioned in a microtome and is resistant to degradationfrom solvents and chemicals used to fix, process and stain tissue, thecassette further including a lid; and an open cell, fully reticulatedfoam material coupled to the tissue cassette, the open cell, fullreticulated foam material configured to engage and retain tissue inplace during processing and embedding, the lid being couplable to thetissue containment portion and used to compress the open cell, fullyreticulated foam against the tissue, and the open cell, full reticulatedfoam material further being capable of successful sectioning in themicrotome and porous to allow infiltration of the solvents and chemicalsused to fix, process and stain tissue, and of embedding material used toembed the tissue while the tissue is retained by the open cell, fullreticulated foam material in the recess.
 2. The histologic tissue samplesupport device of claim 1, wherein the open cell, fully reticulated foammaterial further includes at least one of a polyether or a polyurethane.3. The histologic tissue sample support device of claim 1, wherein theopen cell, fully reticulated foam material is coupled to the lid and isinserted at least partially into the recess upon connecting the lid tothe containment portion.
 4. The histologic tissue sample support deviceof claim 1, wherein the material forming the cassette is at leasttranslucent so as to be non-distracting during tissue analysis.
 5. Thehistologic tissue sample support device of claim 4, wherein the cassetteis formed of a polymer.
 6. The histologic tissue sample support deviceof claim 5, wherein the polymer is a fluorinated polymer.
 7. Thehistologic tissue sample support device of claim 5, wherein the polymeris a fluoropolymer.